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Bacterial Transformation Lab Report

Lab report on Bacterial Tranformation

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Cell Function And Inheritance (BIOL 151)

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St. Cloud State University

Academic year: 2022/2023

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Transformation of the bacterium E. coli using the pGLO plasmid Enkhsolongo Sangibat TA: Nina Orzica Ineza BIOL 151- St. Cloud State University April 17, 2023

Abstract Genetic transformation is one of the most significant scientific discoveries of our time, widely applied in numerous fields such as gene therapy, scientific research, and bioremediation. In this lab experiment, the objective was to perform genetic transformation and determine the success of genetically altering E. coli bacteria using the pGLO plasmid, and to analyze the modified traits expressed by the Green Fluorescent Protein gene. To achieve this goal, we followed a laboratory protocol that involved introducing the plasmid into the bacteria through a series of ice treatments and incubations, followed by transferring the solution to an agar plate for an overnight growth at 37ºC. The experiment aimed to demonstrate genetic transformation of E. coli to express the genetic traits of jellyfish and to glow under UV light using GFP by pGLO genes with arabinose sugar. Two control plates labeled “-” with LB+amp (Plate 1) and LB (Plate 2), and two plates with antibiotics labeled “+” with LB+amp (Plate 3) and LB+amp+ara (Plate 4) were used in the methodology. The hypothesis was that the fully transformed bacteria with Luria Bertani broth would have resistance to ampicillin and could glow bright green under UV light with arabinose and ampicillin present. The results supported the hypothesis by showing that the fully transformed bacteria indeed had resistance to ampicillin and emitted bright green fluorescence under UV light with arabinose and ampicillin. Introduction DNA contains all the genetic information that is necessary for an organism to express various traits through protein synthesis. Genetic transformation is a technique used to express desired genes in an organism and determine its phenotype. This technique has numerous applications in different fields such as agriculture, bioremediation, and medicine. For instance, genetic transformation can be used to treat genetic disorders by replacing the defective gene with a healthy one. In agriculture, plants can be modified to exhibit pest and spoilage resistance. Plasmids, which are small DNA molecules that exist independently of chromosomal DNA, are commonly used to demonstrate genetic transformation.

introducing a gene that codes for Green Fluorescent Protein (GFP). The plates were prepared as follows: “-” on LB plate and LB+amp plate, “+” on another LB+amp and LB+amp+ara plate. First, 250 μl of solution was added to the tubes and 2-3 individual bacteria colonies were added to each tube using sterile techniques and a sterile loop. The DNA plasmid was added with a sterile loop until a visible film was formed into the “+” tube. The tubes were then incubated on ice for 15 minutes along with the tube labelled “-” for initial ice treatment. The tubes were then placed in a 42ºC water bath for exactly 50 seconds for heat shock and put back into the ice for another 2 minutes for second ice treatment. Once that was done, 250 μl of LB broth was added to each tube and incubated for 15 minutes at room temperature for room temperature recovery. The tubes were flicked to mix, and 100 μl from each tube was spread on the agar plates using a sterile loop. The plates were allowed to be incubated at 37ºC until the next day for overnight growth, and then stored at 4ºC until the second lab day. A new sterile loop and sterile pipet was used for each plate and tube. Results The variables of the plates were the presence of arabinose and ampicillin on each plate, and plates were observed for E. coli growth and glow under UV light. The control plates for growth were the LB (-) plate, which didn't show any growth, and the control for glow was the LB+amp (-) plate, which didn't show any glow. The plates with antibiotics, LB+amp (+) and LB+amp+ara (+), showed growth of bacteria colonies, and the plate with arabinose glowed under UV light as green. Although the plate labelled as LB+amp (+) didn't show any growth after the first laboratory experiment, it was repeated again with the same experimental procedure and showed growth in the second attempt.

Discussion The hypothesis for the lab stated that mediums with pGlo will have colonies growing on them and medium with modified arabinose will have glowing bacteria and the results were supporting the hypothesis. As shown in the figures above, the LB (-) control plate did not have any variables added to it and thus had the original colonies on it, but it did not glow under UV light. This confirmed that the UV light did not cause any non-specific fluorescence and that the plates were prepared correctly. The LB+amp (-) plate did not show any bacterial growth, indicating that the ampicillin antibiotic

Figure 1: Visualization of the results of growth under norm LB+amp (+), LB+amp+ara (+) from left to right

Figure 2: Figure 1: Visualization of the results of growth under UV light under conditions LB (-), LB+amp (+), LB+amp+ara (+), LB+amp (-) from left to right

Table 1: Results of the experiment on growth/glow

Plates Growth Fluorescence LB (-) Colonies No LB+amp (-) None No LB+amp (+) Colonies No LB+amp+ara (+) Colonies Yes

Overall, the results of the experiment indicate that the pGlo plasmid successfully transformed into the E. coli genome and that the genes are capable of being expressed. The successful expression of the GFP gene in transformed bacteria confirms that the araC protein does facilitate binding of RNA polymerase and that GFP expression occurs in the presence of arabinose.

Literature Cited Martin Chalfie, Y. Tu, G. Euskirchen, Green Fluorescent Protein as a Marker for Gene Expression. Science 263, 802-805(1994)

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